Kidney disease, a major public health concern in the US and worldwide, is mainly caused by pathological changes in the precise regulation of glomerulus podocyte, the primary filtration unit in the kidney. Dysfunction of podocyte proteins including nephrin leads to renal disease owing to foot processes (FP) retraction, disruption of the filtration barrier and rearrangement of the actin cytoskeleton. However, the mechanisms linking nephrin activation to actin cytoskeleton reorganization are ill understood. The goal of this research project is to identify podocyte proteins involved in the molecular mechanisms controlling the regulation of GTPase, key regulators of the cytoskeletal, activity in space and time during FP development. GTPases act as molecular switches cycling between inactive (GDP-bound) and active (GTP-bound) forms. Several levels of regulation tightly control their activation state and accessibility. GDIs (Guanin Dissociation Inhibitors) function to inhibit GTPase activities. Activation through exchange of GDP for GTP is catalyzed by GEFs (Guanine Exchange Factors) and promotes downstream signaling; GAPs (Guanine- Activating Proteins) accelerate the intrinsic GTPase activity to inactivate the protein and terminate the signal. Given the key roles played by the cytoskeleton in renal podocyte function, the regulation of GTPase activity by GDI, GEFs and GAPs constitute critical points in the regulation of renal cytoskeletal dynamics. Dysregulation of such GTPase cycling can lead to pathological disease states in many cells/tissues. This is particular true in kidney, as evidenced by the dramatic defects in kidney structure and physiology that occur in the GDI knockout mouse, finally leading to death due to renal failure. We hypothesize that nephrin is at the apex of a hierarchy defining compartment identity through the recruitment of GTPases and their regulators. To test our hypothesis, we will pursue the following Aims: Aim1. We will investigate the molecular mechanisms by which a new podocyte GDI binding partner controls GTPase-GDI complexes and its effects on actin cytoskeleton dynamics during nephrin activation. Aim2. We have identified a new binding partner for nephrin that can regulate the inactivation of GTPases. Therefore, we will elucidate by which mechanisms this GAP regulate FP elongation. Aim3: We will determine the spatio-temporal regulation of GTPase-GDI complexes during nephrin stimulation. To this end, we will use a novel and unique live cell GTPase-GDI complex biosensor in in vivo imaging studies, coupled with computational multiplexing analyses. These studies will lead to a greater understanding of how these important components of renal function are controlled, ultimately guiding to unique and increasingly selective therapeutic approaches to intervene in nephritic disorders.